Autoproteolytic stability of a trypsin from the marine crab Cancer pagurus

Autoproteolytic stability is a crucial factor for the application of proteases in biotechnology. In contrast to vertebrate enzymes, trypsins from shrimp and crayfish are known to be resistant against autolysis. We show by characterisation of a novel trypsin from the gastric fluid of the marine crab Cancer pagurus that this property might be assigned to the entire class of crustaceans. The isolated and cloned crab trypsin (C.p.TryIII) exhibits all characteristic properties of crustacean trypsins. However, its overall sequence identity to other trypsins of this systematic class is comparatively low. The high resistance against autoproteolysis was determined by mass spectrometry, which revealed a low susceptibility of the N-terminal domain towards autolysis. By homology modelling of the tertiary structure, the elevated stability was attributed to the distinctly different pattern of autolytic cleavage sites, which is conserved in all known crustacean trypsin sequences.     

Effect of air exposure on nitrogen metabolism in the crab Cancer pagurus.

In C. pagurus exposed to air for 18 h, blood ammonia content decreased within the 2 first hours, then increased at a relatively constant rate (25 microM/h); blood urate content increased at a lower rate (10 microM/h) and a classical blood acidosis was observed. In the cheliped muscle, a transient 22% decrease in GDH activity for ammonia formation and a 48% increase in GDH activity in the reverse reaction (glutamate synthesis) occurred following 6 and 12 h of emersion, respectively. Changes in LDH activity, used as an indicator of anaerobic potential of muscle, were not observed, except for an 18% increase in crabs exposed to air for 24 h. The increase in blood urate content, not known as a response to emersion in decapods, appeared to be different from that observed in response to hypoxia. The relatively low blood ammonia overload and the GDH increased activity for glutamate synthesis suggested that part of the produced ammonia was stored under a bound form in some tissues. The response of C. pagurus to air exposure is discussed on account of the Storey and Storey ('90) theory.     

pH regulation in the stomatogastric ganglion of the crab Cancer pagurus

The regulation of intracellular neuronal pH and pH from the extracellular space was studied in the isolated stomatogastric ganglion of the crab Cancer pagurus. Intracellular neuronal pH was found to be 0.3–0.4 pH units more acidic than the standard bath pH of 7.4 and surprisingly, the extracellular space pH was found to be around 0.1 pH units more alkaline than the bath pH. Extracellular space pH shifts in response to bath pH changes decreased as a function of the depth of the recording site within the ganglion, suggesting the existence of restrictions in the free diffusion of H⁺. The amplitude of these pH_e shifts increased in Na⁺-free saline or with amiloride, suggesting Na⁺-dependent regulation of the extracellular space pH. In Na⁺ free saline or in the presence of amiloride, intracellular pH recovery from an NH₄Cl induced acidosis was reduced, and the H⁺ muffling capacity (cf. Thomas et al. 1991) of the extracellular space was markedly reduced. Changes of bath pH had only small effects on the rhythm generating properties of one of the central pattern generators of the stomatogastric ganglion, while NH₄Cl-induced intraganglionic pH changes markedly altered this rhythm.Abbreviations CPG central pattern generator - ECS extracellular space - LP lateral pyloric neuron - NMDG N-methyl-D-glucamine - PD pyloric dilator neuron - pH_e extracellular space pH - pH_i intracellular pH - pH_o bath pH - STG stomatogastric ganglion - V_ref reference potential     

Efficient identification of proteins from ovaries and hepatopancreas of the unsequenced edible crab,Cancer pagurus,by mass spectrometry and homology-based,cross-species searching

Proteome maps of hepatopancreas (midgut gland) and ovarian tissues of the crustacean, Cancer pagurus (Decapoda; edible crab) have been produced by 2D-PAGE and identification of proteins, following trypsin proteolysis, by electrospray MS/MS and database searching. Owing to the lack of sequence information on proteins and fully sequenced genomes amongst the decapod crustaceans and given the evolutionary distance to the nearest full genome database (Daphnia), it was necessary to adopt a non-conventional identification approach. Thus, a strategy was developed for effective identification of decapod proteins by sequence similarity, homology-based cross-species database searching, using various algorithms and a combination of NCBI Crustacea and Arthropoda databases, together with the Arthropoda PartiGene database (Blaxter, University of Edinburgh). In both hepatopancreas and ovary tissues, the largest group of proteins identified were a variety of enzymes, followed by a smaller number of storage/transport proteins [including vitellogenin (yolk protein), several subunits of hemocyanin, cryptocyanin, ferritin and calreticulin], with fewer structural proteins (actin, tubulin) and heat-shock proteins, in addition to a number of proteins of miscellaneous functions. Such protein identifications allow the development of tools, such as antibodies and RNA/DNA probes, to investigate the functions of the proteins in specific tissues during development.     

In vivo regulation of the mandibular organ in the edible crab, Cancer pagurus

Considerable evidence indicates that methyl farnesoate (MF) production by the crustacean mandibular organs is negatively regulated by neuropeptides from the sinus gland (SG) in the eyestalk. In the crab Cancer pagurus, two neuropeptides (MO-IH-1 and -2) have been isolated from the SG that inhibit MF synthesis by mandibular organs of female crabs in vitro. To test their activity in vivo, we treated eyestalk-ablated male crabs with SG extracts (SGEs) or MO-IH-1 and -2. SGEs reduced haemolymph levels of MF by 60-80%, while MO-IH-1 and -2 had little effect. Protease treatment of SGEs destroyed the in vivo activity, suggesting that the extract contains an additional peptide responsible for the in vivo activity. When separated by reversed-phase high performance liquid chromatography (HPLC), the in vivo activity eluted in fractions prior to MO-IH-1 and -2. When mandibular organs were removed from animals previously treated in vivo with these active fractions, they had reduced levels of MF synthesis and activity of farnesoic acid O-methyl transferase compared with mandibular organs from animals treated with saline. Together, these results indicate that the regulation of the crustacean mandibular organ is complex and may involve several SG compounds. Some of these compounds (i.e., MO-IH-1 and -2) act directly on the tissue while others affect the mandibular organ indirectly.     

A histological study of shell disease syndrome in the edible crab Cancer pagurus.

Shell disease syndrome is characterised by the external manifestation of black spot lesions in the exoskeletons of crustaceans. In the present study, gills, hepatopancreas and hearts from healthy (<0.05% black spot coverage) and diseased (5 to 15% coverage) edible crabs, Cancer pagurus, were examined histologically to determine whether this disease can cause internal damage to such crabs. There was clear evidence of cuticular damage in the gills of diseased crabs leading to the formation of haemocyte plugs termed nodules. Nephrocytes found within the branchial septa of the gills showed an increase in the accumulation of dark material in their vacuoles in response to disease. In the hepatopancreas, various stages of tubular degradation were apparent that correlated with the severity of external disease. Similarly, there was a positive correlation between the number of viable bacteria in the haemolymph and the degree of shell disease severity. Approximately 21% of the haemolymph-isolated bacteria displayed chitinolytic activity. Overall, these findings suggest that shell disease syndrome should not be considered as a disease of the cuticle alone. Furthermore, it shows that in wild populations of crabs shell perforations may lead to limited septicaemia potentially resulting in damage of internal tissues. Whether such natural infections lead to significant fatalities in crabs is still uncertain.     

Deoxyribonucleic acid of the crab Cancer pagurus. 3. Intracellular localization of poly d(A-T) of Cancer pagurus by hybridization in situ

The satellite DNA poly [d(AT) · d(TA)] of the crab Cancer pagurus has been localized in situ by DNA-DNA hybridization in the nuclei of various spermatogenetic, midgut gland, intestinal and tegument cells. The specificity of hybridization was checked by various tests before, during and after hybridization. The nuclear sites revealed by this method were compared with those shown by quinacrine mustard or Giemsa staining. The AT-rich satellite DNA appears to be highly dispersed and does not seem to have any preferential localization inside the crab interphasic nucleus. This situation was compared with that presented by mouse nuclei using similar methods.     

Polymorphic nuclear microsatellite loci for studies of brown crab, Cancer pagurus L

Twelve polymorphic microsatellite DNA loci were isolated from the brown crab, Cancer pagurus L., by construction of microsatellite-enriched genomic libraries. Genotyping of 40 individuals from Norfolk (UK) revealed variable levels of locus polymorphism with an average of 7.75 alleles per locus (range 2-22). The observed and expected heterozygosities per locus ranged from 0.025 to 0.868 and from 0.025 to 0.947, respectively. No evidence of linkage disequilibrium was detected between pairs of loci and genotype proportions at all loci conformed to Hardy-Weinberg equilibrium expectations. The microsatellite loci developed constitute a suite of genetic markers applicable to numerous areas of C. pagurus research.     

Actin in the acrosome of the spermatozoa of the crab,Cancer pagurus L. (decapoda,crustacea)

The cytoskeletal protein actin was identified in the mature spermatozoon of the European edible crab, Cancer pagurus Linnaeus, by indirect immunofluoresce with monoclonal and polyclonal anti-actin antibodies, fluorescent phalloidin, and DNAase I. The actin was localized in two distinct concentric rings within the acrosome vesicle of the spermatozoon and appeared to correlate with the internal zonation of the vehicle. Modifications of the fluorescent pattern for actin were observed in sperm cells which were undergoing changes associated with the acrosome reaction. In these cases, fluorescent staining was observed in the nucleocytoplasm immediately subjacent to the perforatorial column and sometimes in the perforatorial column within the acrosome vesicle. Equally intense fluorescence was observed in an apical perforatorial projection. SDS-PAGE of C. pagurus sperm confirmed the presence of actin in the cells. A single band of actin (approximately 43 kDa) comigrated with rabbit muscle actin when immunoblotted onto nitrocellulose with mouse monoclonal anti-actin. The actin-associated cytoskeletal proteins α-actinin, tropomyosin, and spectrin were also identified within the spermatozoon of C. pagurus using specific polyclonal antibodies, but their presence was not confirmed by SDS-PAGE and immunoblotting. © 1994 Wiley-Liss, Inc.     

Effect of extracellular products of Pseudoalteromonas atlantica on the edible crab Cancer pagurus

Previous studies have shown that injection of extracellular products (ECP) of Pseudoalteromononas atlantica isolated from shell disease-infected edible crabs (Cancer pagurus) into healthy crabs causes rapid death. In this study we examined the nature of the active lethal factor(s) in ECP. Injection of ECP into crabs caused a rapid decline in the total number of circulating hemocytes (blood cells), and the crabs died within 60 to 90 min. The individuals that died showed eyestalk retraction, limb paralysis, and lack of antennal sensitivity, suggesting that the active factor(s) targeted the nervous system. Histopathological investigations showed that affected crabs had large aggregates of hemocytes in the gills, and there was destruction of the tubules in the hepatopancreas. The active factor in ECP was not sensitive to heat treatment (100 degrees C for 30 min) and proteinase K digestion. As lipopolysaccharide (LPS) was a potential candidate for the lethal factor, it was purified from whole P. atlantica bacteria or ECP and subsequently injected into crabs. These crabs had all of the external symptoms observed previously with ECP, such as limb paralysis and eyestalk retraction, and they died within 90 min after challenge, although no significant decline in the number of circulating hemocytes was observed. Similarly, in vitro incubation of hemocytes with purified LPS (1 to 20 microg) from P. atlantica did not result in the clumping reaction observed with ECP but did result in a degranulation reaction and eventual cell lysis. Injection of crabs with Escherichia coli or Pseudomonas aeruginosa LPS (1 microg g of body weight(-1)) did not cause any of the characteristic symptoms observed following exposure to P. atlantica LPS. No mortality of crabs followed the injection of E. coli LPS, but P. aeruginosa LPS caused ca. 80% mortality at 2 h after injection. Overall, these results show that the main virulence factor of P. atlantica for edible crabs is LPS either alone or in combination with other heat-stable factors.     

Claw removal and feeding ability in the edible crab,Cancer pagurus: Implications for fishery practice

Feeding ability and motivation were assessed in the edible crab, Cancer pagurus, to investigate how the fishery practice of de-clawing may affect live crabs returned to the sea. Crabs were either induced to autotomise one claw, or were only handled, before they were offered food. Initially, autotomised and handled crabs were offered mussels, Mytilis edulis, a large part of their natural diet. After 3 days, both autotomised and handled crabs were then offered fish, a more readily handled food source. Autotomy induced crabs consumed significantly fewer mussels and less mussel mass, but ate significantly more mass of fish. This indicates that the effect of autotomy was a reduction of ability to feed on mussels rather than a general reduction of feeding motivation. The discontinuation of claw removal needs to be considered, both for the sustainability of the fishery and animal welfare concerns.     

The specificity of a neutral deoxyribonuclease from Cancer pagurus.

The recently isolated neutral deoxyribonuclease from crab (Cancer pagurus) testes has been characterized in its mode of action and its specificity. The enzyme is a typical endonuclease, forming 5'-phosphate oligonucleotides of large average size; after extensive digestion of calf thymus DNA over 75% of the fragments have a size larger than pentanucleotides and mononucleotides are absent. As far as specificity is concerned, thymidine is very abundant in the 5'-penultimate position (approximately 50%) and in the 3'-terminal position (37%) and almost absent in the 5'-terminal position (approximately 1%), the values quoted concerning Escherichia coli digests of average size (Pn) between 50 and 10.     

Enterospora canceri n. gen., n. sp., intranuclear within the hepatopancreatocytes of the European edible crab Cancer pagurus

Only 1 genus (Nucleospora) within 1 family (Enterocytozoonidae) of the Microsporidia contains species that are parasitic within the nuclei of their host cells; to date, all described intranuclear Nucleospora spp. parasitise fish. This study describes the first intranuclear microsporidian parasite of an invertebrate, the European edible crab Cancer pagurus L. (Decapoda: Cancridae). Infected crabs displayed no obvious external signs, and maximum apparent prevalence of infection within a monthly sample was 3.45%. Infected hepatopancreatic tubules were characterised by varying numbers of hypertrophic and eosinophilic nuclei within epithelial cells. Parasite stages appeared as eosinophilic granular accumulations causing margination of host chromatin. In advanced cases, the tubule epithelia degenerated, with parasites and sloughed epithelial cells appearing in tubule lumens. All life stages of the parasite were observed within host nuclei. Uninucleate meronts were not detected, although binucleate stages were observed. Multinucleate plasmodia (sporogonal plasmodia) contained up to 22 nuclei in section, and late-stage plasmodia contained multiple copies of apparatus resembling the polar filament and anchoring disk, apparently associated with individual plasmodial nuclei. As such, aggregation and early assembly of sporoblast components took place within the intact sporogonial plasmodium, a feature unique to the Enterocytozoonidae. Liberation of sporoblasts from plasmodia or the presence of liberated sporoblasts was not observed in this study. However, large numbers of maturing and mature spores (measuring 1.3 +/- 0.02 x 0.7 +/- 0.01 microm) were frequently observed in direct contact with the host nucleoplasm. Considering the shared features of this parasite with microsporidians of the family Enterocytozoonidae, and the unique presence of this parasite within the nucleoplasm of decapod crustacean hepatopancreatocytes, this parasite (Enterospora canceri) is proposed as the type species of a new genus (Enterospora) of microsporidian. Molecular taxonomic work is now required, comparing Enterospora to Enterocytozoon and Nucleospora, the 2 other genera within the Enterocytozoonidae.     

The isolation of a plasma membrane-rich fraction from the skeletal muscle of two species of marine crab,Carcinus maenas and Cancer pagurus

• 1.1. A rapid method for the isolation of a plasma membrane-rich fraction from crab leg muscle, with high purity and yield recovery was developed.
• 2.2. The method is based on sodium iodide extraction of the crude homogenate, followed by centrifugation on Percoll self-creating gradient.
• 3.3. (Na⁺K⁺)ATPase and alkaline phosphodiesterase I were used as marker enzymes for the plasma membrane and revealed levels of purification of approximately 13-fold and yields recovery of the total activity in the crude muscle homogenate of approximately 18%, for both species of crab studied.

     

The partition of copper and cadmium between different charge-forms of metallothionein in the digestive tubules of the crab, Cancer pagurus

In the presence of 2-mercaptoethanol DEAE cellulose chromatography of desalted hepatopancreas cytosols of crabs rich in both copper and cadmium yielded three peaks, probably all due to metallothioneins, with very different copper to cadmium ratios. Addition of a copper glycine complex to a desalted crab cytosol containing cadmium metallothionein gave a chromatogram similar to that obtained by in vivo administration of copper and cadmium. The chromatographic distribution of the metals between the different charge-forms in the distal and proximal portions of the tubules was basically similar although the concentrations of the metals were different.